CN109628345B

CN109628345B - Composite microbial inoculum for preventing and treating brown rot of succulent plants as well as preparation method and application of composite microbial inoculum

Info

Publication number: CN109628345B
Application number: CN201811628263.1A
Authority: CN
Inventors: 赵钢勇; 刘金龙; 顾欣燕; 刘刚; 肖培英; 赵晓琛
Original assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Current assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2021-07-09
Anticipated expiration: 2038-12-28
Also published as: CN109628345A

Abstract

The invention relates to a composite microbial inoculum for preventing and treating succulent plant brown rot, a preparation method and application thereof, wherein the composite microbial inoculum is prepared from Bacillus Pumilus Fermentation product of MES828, Brevibacillus laterosporus and (3) fully mixing the fermentation products of MES818 according to the volume ratio of 1-4:1-4, centrifuging at 5000-10000 rpm for 15-30 min to obtain thalli, and suspending and scattering the thalli by using sterile water to prepare the culture. The composite microbial inoculum for preventing and treating brown rot of the polypide of Crassulaceae can effectively prevent and treat brown rot of the polypide of Crassulaceae, can promote nutrient absorption and utilization of the polypide of Crassulaceae, improves the form of the polypide of Crassulaceae, and has important positive significance for enhancing the ornamental value of the polypide of Crassulaceae.

Description

Composite microbial inoculum for preventing and treating brown rot of succulent plants as well as preparation method and application of composite microbial inoculum

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a composite microbial inoculum for preventing and treating succulent plant brown rot, and a preparation method and application thereof.

Background

Succulent plants, also known as succulent plants or succulent plants. The color is rich, the body is full, the texture is various, the adaptability is strong, and the like, so the color is popular with the public consumers. With the development of economy and the progress of society, the consumption concept of people is greatly changed, more and more people begin to pursue high-quality life, the sales volume of the crassulaceae succulent plants is promoted to be increased rapidly, the incidence rate of brown rot of the crassulaceae succulent plants is increased year after year and is increasingly prominent, the income of growers is seriously influenced, and the whole industry is seriously influenced. Therefore, the control of the harm of the polypide plants in the Crassulaceae becomes a problem to be solved urgently in production.

Brown rot is one of fungal diseases. The brown rot has very strong adaptability and viability, can not only take nutrition from living plant tissues, but also survive in dead residues. The disease-causing capability of the brown rot is super strong, once the conditions are proper, the brown rot can invade into succulent plant organs of the sedum polytrichaceae and can be rapidly propagated. The leaf is infected with diseases, is yellow or tawny, and gradually expands into round or oval scab and is reddish brown; when the petals are damaged, the petals are initially in water stain-like brown spots and gradually expand, and the whole petals become withered, withered and drooping; the bulb is damaged and irregular black spots are generated on the appearance. Generating a grey mould layer on the infected stems and leaves under a humid condition; within the leaf sheath, black sclerotia are produced on the surface of the bulb or in the soil. At present, the chemical control is mainly used for controlling brown rot in the production of the varieties of succulent plants of Crassulaceae without brown rot resistance. The chemical control causes the drug resistance of pathogenic bacteria, is difficult to effectively prevent the reoccurrence of diseases from the root, has larger environmental pollution and harms the ecological balance, and the adoption of microorganisms to control plant diseases is one of the most effective biocontrol means, but no relevant report exists on the research of the biocontrol microbial inoculum on the brown rot of the polypide plants in the Crassulaceae.

Disclosure of Invention

The invention aims to solve the technical problem of a composite microbial inoculum for preventing and treating the brown rot of succulent plants and a preparation method and application thereof. The compound microbial inoculum for preventing and treating brown rot of succulent plants, prepared by the invention, can effectively prevent and treat brown rot of succulent plants in Crassulaceae, can promote absorption and utilization of nutrients by the succulent plants in Crassulaceae, improves the shapes of the succulent plants in Crassulaceae, and has important positive significance for enhancing the ornamental value of the succulent plants in Crassulaceae.

In order to solve the problems, the invention adopts the following technical scheme:

the invention provides a preparation method of a composite microbial inoculum for preventing and treating succulent plant brown rot, which is prepared by fully mixing a fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and a fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 according to a volume ratio of 1-4:1-4, then centrifuging at 5000-10000 rpm for 15-30 min to obtain thalli, and carrying out resuspension and scattering on the thalli by using sterile water, so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

The Bacillus Pumilus (Bacillus Pumilus) MES828 fermentation product and the Bacillus laterosporus (Brevibacillus laterosporus) MES818 fermentation product are both prepared by adopting a liquid high-density fermentation technology; wherein the viable count of Bacillus Pumilus (Bacillus Pumilus) MES828 fermentation product is not less than 2.5 × 10⁹cFu/g, the number of viable bacteria in the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 is not less than 2.5 × 10⁹cFu/g。

A bacterial strain for preventing and treating brown rot of succulent plants in Crassulaceae is Bacillus Pumilus MES828 which is preserved in China general microbiological culture Collection center with the preservation address of No. 3 Xilu No.1 of Beijing Kogyo-Yang area, the preservation date of 2018, 12 months 03 days and the preservation number of CGMCC No. 16858.

The Brevibacillus laterosporus MES818 is preserved in the China general microbiological culture collection center, the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, the preservation date is 2018, 12 and 03 days, and the preservation number is CGMCC No. 16859.

The bacillus pumilus is in a thin rod shape, is gram-positive, has the size of 0.6-0.7 mu m multiplied by 2.0-3.0 mu m, has an oval spore shape, is a midlife or a near-midlife spore, does not expand the spore significantly, and has a circular colony, a faint yellow colony, opacity, flatness, a moist surface and regular edges on a nutrient agar culture medium. Positive reaction: contacting with an enzyme; an oxidase; V-P determination; liquefying gelatin; citrate utilization. Negative reaction: utilizing propionate; hydrolyzing starch; nitrate reduction; liquefying gelatin; and (4) anaerobic growth. The specific primers are adopted for multiplex PCR amplification, the strain generates a unique amplification product, and the size of a strip is the same as that of Bacillus Pumilus (Bacillus Pumilus).

The Brevibacillus laterosporus thallus is in a thin rod shape and is gram-positive, the size is 1.0-1.2 mu m multiplied by 2.5-3.0 mu m, the Duzhou shaped laterosporus is provided, the thallus is oval, cysts are expanded, and the colony is round, small, grey white, neat in edge, moist and smooth in surface and semitransparent on a nutrient agar culture medium. Positive reaction: contacting with an enzyme; an oxidase; liquefying gelatin; nitrate reduction; and (4) anaerobic growth. Negative reaction: V-P determination; utilizing citrate; and (3) hydrolyzing the starch. The strain produces a unique amplification product by performing multiple PCR amplification by using specific primers, and the size of a band is the same as that of Brevibacillus laterosporus (Brevibacillus laterosporus).

Preferably, the high-density fermentation method of the bacillus pumilus comprises the following steps:

(1) preparing strains; transferring separated Bacillus Pumilus (Bacillus Pumilus) MES828 to an eggplant bottle culture medium, culturing at 35-37 ℃ for 48-72 h to obtain an activated strain, adding 30ml of a protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 5-15%, and culturing at 35-37 ℃ at 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation according to the inoculation amount of 5-15%, wherein the charging coefficient of the seed tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotating speed is 220-250 rpm, the tank pressure is kept between 0.05-0.08 MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.5-7.0, the fermentation period is 24-36 h, and 5-10M NaOH is fed in the fermentation process to control the pH value of a fermentation liquid to be 6.5-7.0;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 5-15% for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotating speed is 220-250 rpm, the tank pressure is kept between 0.05MPa and 0.08MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.5-7.0, when the fermentation is carried out for 24-36 h, an external nutrient (a mixed solution of 0.5-1% of glucose and 1-4% of soybean polypeptide) is fed in a feeding mode, continuing the fermentation for 10-12 h, and after the fermentation is finished, obtaining a Bacillus pumilus MES828 liquid fermentation product.

Preferably, the high-density fermentation method of the brevibacillus laterosporus comprises the following steps:

(1) preparing strains; transferring separated Brevibacillus laterosporus MES818 to an eggplant bottle culture medium, culturing at 35-37 ℃ for 48-72 h to obtain an activated strain, adding 30ml of protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(4) fermentation in a fermentation tank: after the fermentation of the seed tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 5-15% for liquid fermentation, wherein the charging coefficient of the fermentation tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotation speed is 220-250 rpm, the tank pressure is kept between 0.05-0.08 MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation medium is 6.5-7.0, when the fermentation is carried out for 24-36 h, an external nutrient (a mixed solution of 0.5-1% of glucose and 1-4% of soybean polypeptide) is added in a feeding mode, the fermentation is continued for 10-12 h, and a brevibacillus laterosporus MES818 liquid fermentation product can be obtained after the fermentation is finished.

Preferably, the eggplant bottle culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 0.5-2% of tryptone, 0.5-2% of beef extract, 0.2-1% of sodium chloride, 1.5-2% of agar and the balance of deionized water, controlling the pH value to be 7.0-7.3, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium for the Bacillus pumilus MES828 during culture specifically comprises the following components in parts by mass: 0.5-2% of fish peptone, 0.5-2% of soybean peptone, 0.5-2% of yeast powder, 0.2-1% of NaCl, natural pH value and sterilization at 121 ℃ for 30 min;

the seed tank culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 2 to 4 percent of molasses, 0.5 to 3 percent of soybean meal, 0.5 to 2 percent of enzymolysis peanut meal, 0.5 to 1 percent of yeast extract, 0.2 to 2 percent of sodium chloride, 0.5 to 2 thousandths of magnesium sulfate, 0.2 to 2 percent of citric acid, KH₂PO₄0.05 to 0.10 percent of the total weight of the mixture and the balance of deionized water, controlling the pH value to be 7.0 to 7.3, and sterilizing the mixture for 30min at the temperature of 121 ℃;

the enzymatic peanut meal is obtained by compounding flavourzyme and neutral protease according to the ratio of 1: 3-3: 1, adding 0.5-1% (E/S, calculated by the mass of a substrate), carrying out enzymatic hydrolysis at the temperature of 40-55 ℃ for 4-6 h, carrying out vacuum filtration, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium for the bacillus pumilus MES828 during culture specifically comprises the following components in percentage by mass: 1 to 3 percent of white sugar, 1 to 3 percent of molasses, 0.5 to 2 percent of peanut cake powder, 0.5 to 1 percent of yeast extract, 0.2 to 2 percent of sodium chloride, 0.5 to 2 thousandths of magnesium sulfate, 0.2 to 2 percent of citric acid and KH₂PO₄0.05-0.1 percent of neutral protease, 0.5-2 per mill of flavourzyme, 0.2-1.5 per mill of foam killer and the balance of deionized water, wherein the pH value of a culture medium is 6.0-7.0, and the culture medium is sterilized for 30min at 121 ℃.

Preferably, the eggplant flask culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 0.5-2% of tryptone, 0.5-2% of beef extract, 0.2-1% of sodium chloride, 1.5-2% of agar and the balance of deionized water, controlling the pH value to be 7.0-7.3, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 0.5-2% of fish peptone, 0.5-2% of soybean peptone, 0.5-2% of yeast powder, 0.2-1% of NaCl, natural pH value and sterilization at 121 ℃ for 30 min;

the seed tank culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 0.5-2% of yeast extract powder, 1-3% of molasses, 1-4% of enzymatic hydrolysis peanut meal powder, 0.5-1% of monopotassium phosphate, 0.03-0.05% of magnesium sulfate, 0.03-0.05% of calcium carbonate, 0.5-1% of sodium chloride and the balance of deionized water, wherein the pH is natural, and the mixture is sterilized at 121 ℃ for 30 min;

the fermentation tank liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 2-4% of glucose, 1-3% of molasses, 1-3% of yeast extract powder, 2-4% of peanut meal, 1-3% of sodium chloride, 1-2% of magnesium sulfate, 0.5-2% of neutral protease, 0.5-2% of flavourzyme, 0.5-3% of sodium citrate, 0.2-1.5% of foam killer and the balance of deionized water, wherein the pH value of the culture medium is 6.0-7.0, and the culture medium is sterilized at 121 ℃ for 30 min.

The invention also provides application of the compound microbial inoculum for preventing and treating the brown rot of the succulent plants, which is characterized in that the compound microbial inoculum is diluted by water according to the ratio of 1: 1000-1: 500, and then the compound microbial inoculum is applied to the succulent plant leaves in a leaf surface spraying mode or applied to the roots of the succulent plants in a root irrigation mode.

Advantageous effects

The effect test of the composite microbial inoculum for preventing and treating the brown rot of the succulent plants of the Crassulaceae shows that the preventing and treating efficiency of the brown rot pathogenic bacteria of the succulent plants of the Crassulaceae reaches over 84 percent.

The compound microbial inoculum for preventing and treating the sedum succulent brown rot can promote nutrient absorption and utilization of sedum succulent plants, improve the form of the sedum succulent plants and have important positive significance for enhancing the ornamental value of the sedum succulent plants.

The composite microbial inoculum for preventing and treating the stonecrop succulent brown rot has the viable count of more than 25 hundred million/mL, stable microbial activity and pH of 4.0-7.5.

The compound microbial inoculum for preventing and treating the brown rot of the polypide of the crassulaceae is compounded by adopting multiple strains, can maintain the balance of root microbial flora of the polypide of the crassulaceae and improve the structure of rhizosphere microbial flora.

According to the composite microbial inoculum for preventing and treating the brown rot of the polypide in the Crassulaceae, in the strain fermentation process, the culture medium of the seeding tank adopts the enzymolyzed peanut meal as one of raw materials, and the enzymolyzed peanut meal is rich in a large amount of soluble proteins, small molecular polypeptides and other substances, so that the rapid utilization of strains is facilitated, and the growth cycle is shortened.

Compared with a single bacillus pumilus and a single bacillus brevis strain, the composite microbial inoculum for preventing and treating the brown rot of the polyporus plants in the Crassulaceae family has stronger antagonistic action, and the two compounded strains are mutually synergistic, can be fixedly planted, propagated and transferred in roots, root tables and bodies of the polyporus plants in the Crassulaceae family, has stronger antagonistic effect on the brown rot of the polyporus plants in the Crassulaceae family, and has larger application potential in preventing and treating the brown rot.

Drawings

FIG. 1 is a photograph showing colonies and cells of MES828 Bacillus pumilus;

FIG. 2 is a photograph showing colonies and cells of MES818 Brevibacillus laterosporus.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

A bacterial strain for preventing and treating brown rot of succulent plants in Crassulaceae comprises Bacillus Pumilus (Bacillus Pumilus) MES828, which is preserved in China general microorganism strain preservation management center at No. 3 of Beijing Shang Naoyang district North Xilu No.1, wherein the preservation date is 2018, 12 months 03 and the preservation number is CGMCC No. 16858. The colony and the photograph of the cell are shown in FIG. 1.

The strain is Brevibacillus laterosporus MES818 and is preserved in China general microbiological culture collection center, the preservation address is No. 3 Xilu No.1 Beijing Korean district, the preservation date is 2018, 12 and 03 days, and the preservation number is CGMCC No. 16859. The colony and the photograph of the cell are shown in FIG. 2.

The antagonistic effect test of the prepared bacillus pumilus and bacillus laterosporus on the brown rot of the succulent plants in the crassulaceae comprises the following steps:

the pathogenic bacteria of the brown rot of the tested succulent plants in Crassulaceae are collected from Vietnamese farmhouse bases in Chengjizhen Mei stone highway in Wuqing district.

Preparation of pathogenic bacteria of brown rot of succulent plants of Crassulaceae:

clamping diseased succulent leaves by using sterile tweezers, immersing 5mm succulent leaves into 75% alcohol for 2-3 seconds, transferring the succulent leaves into a 3% sodium hypochlorite solution for treatment for 3-5 min, putting the solution into sterile water for changing and washing for 3 times, 2-3 min each time, then sucking surface moisture by using sterile filter paper, finally putting leaf tissues on a PDA culture medium flat plate, culturing for 3-5 days at 28 ℃, picking a little of lawn and streaking after hypha or spore grows out, transferring the lawn to a PDA slant culture medium for culture, and culturing for later use at 28 ℃ for 3-6 days.

Pathogenicity study:

adding 15ml of sterile water into a test tube with a grown pathogenic bacteria slant, scraping lawn to obtain bacterial suspension, taking a healthy Crassulaceae succulent plant, slightly cutting off the leaves of the Crassulaceae succulent plant with an inoculating needle, inoculating the pathogenic bacteria, and inoculating the sterile water as a control group. And (4) performing routine management in a greenhouse, inoculating 5 succulent plants in each treatment, repeating each treatment for 5 times, and observing the morbidity. The results show that disease symptoms caused by inoculating the isolate into healthy crassulaceae succulent plant leaves are similar to the disease crassulaceae succulent plant symptoms, brown soft rot appears, the whole leaf is rotten at the later stage of culture, the pathogenicity determination is strong, and the isolate is identified as Pantoea ananatis (Pantoea ananatis) through the identification of a strain detection mechanism of the department of agriculture, and the number of the isolate is KHSWBYJ-012.

Functional detection

Plate confrontation tests of bacillus pumilus MES828 and bacillus laterosporus MES 818:

selecting separated and purified Crassulaceae succulent plant brown rot fungus moss by using an inoculating loop, smearing the Crassulaceae succulent plant brown rot fungus moss in the center of a sterile nutrient agar plate, respectively inoculating Bacillus pumilus MES828 and Bacillus brevis MES818 which are cultured to logarithmic phase at a position 2.0cm away from an indicator fungus piece, and setting a blank control. After the culture at 30 ℃ for 4 days, 2 kinds of bacillus can obviously inhibit the growth of pathogenic bacteria, and a wider antibacterial zone appears. Culturing at constant temperature of 30 ℃, measuring the growth amount (colony radius) of a control group and the growth amount (growth inhibition radius after MES828 and MES818 are inoculated) of brown rot pathogenic bacteria of a polypide plant of Crassulaceae when a blank control is about to grow over the whole culture dish, and expressing the inhibition effect by the antagonistic action bacteriostasis rate (bacteriostasis rate, the growth amount of the control group-the growth amount of the experiment group)/the growth amount of the control group multiplied by 100 percent), which is specifically shown in the following table 1.

TABLE 1 results of antagonistic tests of Bacillus pumilus MES828 and Brevibacillus laterosporus MES818 against brown rot of a succulent plant of Crassulaceae.

The experimental results show that: the results in table 1 show that the inhibition rate of the Bacillus pumilus MES828 on the brown rot of the succulent plants in the Crassulaceae family reaches 85.7 percent, and the transparent bacteriostatic bandwidth is 10.3 mm; the inhibition rate of Brevibacillus laterosporus MES818 on the brown rot of the polypide plant in the Crassulaceae is 85.4 percent, and the transparent inhibition bandwidth is 10.0 mm. The bacillus pumilus MES828 and the bacillus laterosporus MES818 have obvious inhibition effect on the brown rot of the polypide plants in the crassulaceae, and have biological control potential for preventing and treating the brown rot of the polypide plants in the crassulaceae.

Example 2

The invention also provides a composite microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae, which is prepared by fully mixing the fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 according to the volume ratio of 1:4, then centrifuging the mixture for 30min at 5000rpm to obtain thalli, and resuspending and scattering the thalli by using sterile water, so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～1.0×10¹⁰Within the range.

The Bacillus Pumilus MES828 fermentation product and the Bacillus laterosporus MES818 fermentation product are both prepared by adopting a liquid high-density fermentation technology, wherein the viable count of the Bacillus Pumilus (Bacillus Pumilus) MES828 fermentation product is not less than 2.5 multiplied by 10⁹cFu/g; the number of viable bacteria in the fermentation product of Brevibacillus laterosporus MES818 is not less than 2.5 × 10⁹cFu/g。

(1) preparing strains; transferring separated Bacillus Pumilus (Bacillus Pumilus) MES828 to an eggplant bottle for culturing, culturing at 37 ℃ for 72 hours to obtain an activated strain, adding 30ml of a protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into the eggplant bottle, scraping by using an aseptic spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 5%, and culturing at 37 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank with the inoculation amount of 10% for fermentation, wherein the charging coefficient of the seed tank is 45%, the fermentation temperature is 37 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1:1, the initial pH value of a seed culture medium is 7.0, the fermentation period is 36h, and 5M NaOH is fed during the fermentation process to control the pH value of the fermentation liquid to be 6.8;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to 15% of inoculation amount for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 50%, the fermentation temperature is 37 ℃, the rotation speed is 250rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1:0.5, the initial pH value of a fermentation culture medium is 7.0, after the fermentation is carried out for 24 hours, feeding exogenous nutrients (mixed liquid of 0.5% of glucose and 1% of soybean polypeptide) in a feeding manner, continuing the fermentation for 12 hours, and after the fermentation is finished, obtaining a Bacillus pumilus MES828 liquid fermentation product.

(1) preparing strains; transferring the separated Brevibacillus laterosporus MES818 strain to an eggplant bottle culture medium, culturing at 35 ℃ for 72 hours to obtain an activated strain, adding 30ml of protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 10%, and culturing at 35 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with 15% of inoculation amount, wherein the seed tank charging coefficient is 50%, the fermentation temperature is 35 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 7.0, the fermentation period is 36h, and 5M NaOH is fed during the fermentation process to control the pH value of the fermentation liquid to be 6.8;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to 15% of inoculation amount for liquid fermentation, wherein the charging coefficient of the fermentation tank is 55%, the fermentation temperature is 35 ℃, the rotation speed is 250rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1:1, the initial pH value of a fermentation culture medium is 7.0, after the fermentation is carried out for 36h, feeding exogenous nutrients (1% glucose solution and 1% soybean polypeptide mixed solution) in a feeding manner, continuing the fermentation for 10h, and after the fermentation is finished, obtaining a brevibacillus laterosporus MES818 liquid fermentation product.

Preferably, the eggplant bottle culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 1% of tryptone, 0.5% of beef extract, 0.5% of sodium chloride, 2% of agar and the balance of deionized water, controlling the pH value at 7.0, and sterilizing at 121 ℃ for 30 min;

the seed liquid culture medium for the Bacillus pumilus MES828 during culture specifically comprises the following components in parts by mass: 2% of fish peptone, 0.5% of soybean peptone, 1% of yeast powder, 0.5% of NaCl, natural pH value, and sterilizing at 121 ℃ for 30 min;

the seed tank culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: molasses 3%, soybean powder 0.5%, enzymolyzed peanut cake powder 2%, yeast extract 1%, sodium chloride 0.3%, magnesium sulfate 2 ‰, citric acid 0.5%, KH₂PO₄0.10 percent of deionized water, the balance, the pH value is controlled at 7.0, and the sterilization is carried out for 30min at the temperature of 121 ℃;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to a ratio of 1:3, adding 1% (E/S by mass of a substrate) of enzyme, carrying out enzymolysis at 40 ℃ for 6h, carrying out vacuum filtration, and drying at 80 ℃;

the fermentation tank liquid culture medium for the bacillus pumilus MES828 during culture specifically comprises the following components in percentage by mass: white sugar 2%, molasses 2%, peanut cake powder 2%, yeast extract 0.5%, sodium chloride 0.5%, magnesium sulfate 0.5 ‰, citric acid 0.5%, KH₂PO₄0.1 percent, 2 per mill of neutral protease, 2 per mill of flavourzyme, 0.5 per mill of natural enemy and the balance of deionized water, the pH value of a culture medium is 7.0, and the sterilization is carried out for 30min at 121 ℃.

Preferably, the eggplant flask culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 1% of tryptone, 1% of beef extract, 0.5% of sodium chloride, 2% of agar and the balance of deionized water, controlling the pH value to be 7.0, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 2% of fish peptone, 0.5% of soybean peptone, 1% of yeast powder, 0.5% of NaCl, natural pH value, and sterilizing at 121 ℃ for 30 min;

the seed tank culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 1% of yeast extract powder, 2% of molasses, 4% of enzymatic peanut meal, 0.5% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.05% of calcium carbonate, 0.5% of sodium chloride and the balance of deionized water, wherein the pH value is natural, and the sterilization is carried out for 30min at 121 ℃;

the enzymatic peanut meal is obtained by compounding flavourzyme and neutral protease according to the ratio of 1:1, adding 1% (E/S by mass of a substrate) of enzyme, carrying out vacuum filtration on the peanut meal for 4 hours at the enzymatic hydrolysis temperature of 50 ℃ and drying the peanut meal at the temperature of 80 ℃;

the fermentation tank liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 3% of glucose, 2% of molasses, 1% of yeast extract powder, 4% of peanut meal, 1% of sodium chloride, 1% of magnesium sulfate, 2% of neutral protease, 2% of flavourzyme, 1% of sodium citrate, 0.5% of foam killer and the balance of deionized water, wherein the pH value of a culture medium is 7.0, and the culture medium is sterilized for 30min at 121 ℃.

Example 3

The invention also provides a composite microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae, which is prepared by fully mixing the fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 according to the volume ratio of 3:2, centrifuging at 8000rpm for 20min to obtain thalli, and resuspending the thalli by using sterile water to obtain the composite microbial inoculum, so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

The Bacillus pumilus MES828 liquid fermentation product and the short sporeThe Bacillus MES818 fermentation product is prepared by liquid high density fermentation technology, wherein the viable count of Bacillus Pumilus MES828 fermentation product is not less than 2.5 × 10⁹cFu/g; the number of viable bacteria in the fermentation product of Brevibacillus laterosporus MES818 is not less than 2.5 × 10⁹cFu/g。

(1) preparing strains; transferring separated Bacillus Pumilus (Bacillus Pumilus) MES828 to an eggplant bottle culture medium, culturing at 35 ℃ for 48h to obtain an activated strain, adding 30ml of a protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing in a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 15%, and culturing at 35 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with 15% of inoculation amount, wherein the seed tank charging coefficient is 55%, the fermentation temperature is 35 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.08Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.8, the fermentation period is 24 hours, and 5M NaOH is fed during the fermentation process to control the pH value of the fermentation liquid to be 7.0;

(4) fermentation in a fermentation tank: after the fermentation of the seed tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 10% for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 45%, the fermentation temperature is 35 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.08MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.8, the fermentation is carried out for 36 hours, a culture medium (a mixed solution of 1% of glucose and 3% of soybean polypeptide) is fed in a feeding manner, the fermentation is continued for 10 hours, and after the fermentation is finished, a Bacillus pumilus MES828 liquid fermentation product can be obtained.

(1) preparing strains; transferring the separated Brevibacillus laterosporus MES818 strain to an eggplant bottle culture medium, culturing at 37 ℃ for 48h to obtain an activated strain, adding 30ml of protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculation amount of 8%, and culturing at 36 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank by an inoculation amount of 5% for fermentation, wherein the charging coefficient of the seed tank is 45%, the fermentation temperature is 37 ℃, the rotation speed is 250rpm, the tank pressure is kept between 0.08Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.5, the fermentation period is 24 hours, and 10M NaOH is fed during the fermentation process to control the pH value of a fermentation liquid to be 7.0;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 10% for liquid fermentation, wherein the charging coefficient of the fermentation tank is 45%, the fermentation temperature is 37 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.5, the fermentation is carried out for 24h, the culture medium (mixed liquid of 0.5% glucose and 1% soybean polypeptide) is fed in a feeding manner, the fermentation is continued for 12h, and after the fermentation is finished, a short bacillus laterosporus MES818 liquid fermentation product can be obtained.

Preferably, the eggplant bottle culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 2% of tryptone, 2% of beef extract, 1% of sodium chloride, 1.5% of agar and the balance of deionized water, controlling the pH value to be 7.3, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium for the Bacillus pumilus MES828 during culture specifically comprises the following components in parts by mass: fish peptone 0.5%, soybean peptone 2%, yeast powder 2%, NaCl 0.5%, natural pH value, sterilizing at 121 deg.C for 30 min;

the seed tank culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: molasses 2%, soybean3% of flour, 1% of enzymolyzed peanut meal, 0.5% of yeast extract, 0.2% of sodium chloride, 1% of magnesium sulfate, 0.2% of citric acid and KH₂PO₄0.05 percent and the balance of deionized water, controlling the pH value to be 7.3, and sterilizing for 30min at 121 ℃;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to the ratio of 1:1, adding 0.5% (E/S, calculated by the mass of a substrate) of enzyme, carrying out enzymolysis at the temperature of 50 ℃ for 4 hours, carrying out vacuum filtration, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium for the bacillus pumilus MES828 during culture specifically comprises the following components in percentage by mass: 1% of white sugar, 3% of molasses, 1% of peanut cake powder, 1% of yeast extract, 2% of sodium chloride, 1% of magnesium sulfate, 0.2% of citric acid and KH₂PO₄0.05%, 1 per mill of neutral protease, 2 per mill of flavourzyme, 1.5 per mill of natural enemy and the balance of deionized water, the pH value of a culture medium is 6.5, and the culture medium is sterilized for 30min at 121 ℃.

Preferably, the eggplant flask culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 2% of tryptone, 0.5% of beef extract, 0.2% of sodium chloride, 1.5% of agar and the balance of deionized water, controlling the pH value to be 7.3, and sterilizing at 121 ℃ for 30 min;

the seed liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: sterilizing fish peptone 0.5%, soybean peptone 2%, yeast powder 0.5%, NaCl 1%, natural pH at 121 deg.C for 30 min;

the seed tank culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 2% of yeast extract powder, 3% of molasses, 2% of enzymatic peanut meal powder, 1% of monopotassium phosphate, 0.03% of magnesium sulfate, 0.03% of calcium carbonate, 1% of sodium chloride and the balance of deionized water, wherein the pH is natural, and the sterilization is carried out at 121 ℃ for 30 min;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to a ratio of 1:3, adding 0.5% (E/S, calculated by the mass of a substrate) of enzyme, carrying out enzymolysis at 55 ℃ for 4 hours, carrying out vacuum filtration, and drying at 80 ℃;

the fermentation tank liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 2% of glucose, 1% of molasses, 3% of yeast extract powder, 2% of peanut meal, 3% of sodium chloride, 2% of magnesium sulfate, 2% of neutral protease, 0.5% of flavourzyme, 0.5% of sodium citrate, 1.5% of natural enemy and the balance of deionized water, wherein the pH value of a culture medium is 6.8, and the culture medium is sterilized for 30min at 121 ℃.

Example 4

The invention also provides a composite microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae, which is prepared by fully mixing the fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 according to the volume ratio of 4:1, centrifuging the mixture for 15min at 10000rpm to obtain thalli, and suspending and scattering the thalli by sterile water, so that the total number of the living bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

(1) preparing strains; transferring separated Bacillus Pumilus (Bacillus Pumilus) MES828 to an eggplant bottle for culturing at 36 ℃ for 60 hours to obtain an activated strain, adding 30ml of a protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into the eggplant bottle, scraping by using an aseptic spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 10%, and culturing at 36 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with the inoculation amount of 5%, wherein the charging coefficient of the seed tank is 50%, the fermentation temperature is 36 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.07Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.5, the fermentation period is 30h, and 5M NaOH is fed during the fermentation process to control the pH value of a fermentation liquid to be 6.5;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 5% for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 55%, the fermentation temperature is 36 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.07Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.5, the fermentation is carried out for 30h, exogenous nutrients (mixed liquid of 0.8% of glucose and 2% of soybean polypeptide) are fed in a feeding mode, the fermentation is continued for 11h, and after the fermentation is finished, a Bacillus pumilus MES828 liquid fermentation product can be obtained.

(1) preparing strains; transferring separated Brevibacillus laterosporus MES818 to an eggplant bottle culture medium, culturing at 36 ℃ for 63h to obtain an activated strain, adding 30ml of protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 15%, and culturing at 36 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with the inoculation amount of 10%, wherein the charging coefficient of the seed tank is 55%, the fermentation temperature is 36 ℃, the rotating speed is 240rpm, the tank pressure is kept between 0.07Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.8, the fermentation period is 33h, and 5M NaOH is fed during the fermentation process to control the pH value of a fermentation liquid to be 6.5;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 5% for liquid fermentation, wherein the charging coefficient of the fermentation tank is 50%, the fermentation temperature is 36 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.08MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.8, when the fermentation is carried out for 30 hours, exogenous nutrients (mixed liquid of 0.8% of glucose and 4% of soybean polypeptide) are fed in a feeding mode, the fermentation is continued for 11 hours, and after the fermentation is finished, a liquid fermentation product of Brevibacillus laterosporus MES818 can be obtained.

Preferably, the eggplant bottle culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: tryptone 0.5%, beef extract 1%, sodium chloride 0.2%, agar 1.7%, and deionized water in balance, controlling pH at 7.2, and sterilizing at 121 deg.C for 30 min;

the seed liquid culture medium for the Bacillus pumilus MES828 during culture specifically comprises the following components in parts by mass: sterilizing fish peptone 1%, soybean peptone 1%, yeast powder 0.5%, NaCl 2%, natural pH value at 121 deg.C for 30 min;

the seed tank culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: molasses 4%, soybean powder 2%, enzymolyzed peanut meal 0.5%, yeast extract 0.8%, sodium chloride 2%, magnesium sulfate 0.5 ‰, citric acid 2%, KH₂PO₄0.08 percent of the total weight of the mixture, and the balance of deionized water, controlling the pH value to be 7.2, and sterilizing for 30min at 121 ℃;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to the ratio of 3:1, adding 0.8% (E/S, calculated by the mass of a substrate) of enzyme, carrying out enzymolysis at the temperature of 55 ℃ for 6h, carrying out vacuum filtration, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium for the bacillus pumilus MES828 during culture specifically comprises the following components in percentage by mass: 3% of white sugar, 1% of molasses, 0.5% of peanut cake powder, 0.8% of yeast extract, 0.2% of sodium chloride, 2% of magnesium sulfate, 2% of citric acid and KH₂PO₄0.08%, 0.5% of neutral protease, 0.5% of flavourzyme, 0.2% of natural killer and the balance of deionized water, wherein the pH value of the culture medium is 6.0, and the culture medium is sterilized for 30min at 121 ℃.

Preferably, the eggplant flask culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: tryptone 0.5%, beef extract 2%, sodium chloride 1%, agar 1.7%, and deionized water in balance, controlling pH at 7.2, and sterilizing at 121 deg.C for 30 min;

the seed liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 1% of fish peptone, 1% of soybean peptone, 2% of yeast powder, 0.2% of NaCl, natural pH value, and sterilizing at 121 ℃ for 30 min;

the seed tank culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 0.5% of yeast extract powder, 1% of molasses, 1% of enzymatic peanut meal, 0.7% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.05% of calcium carbonate, 1% of sodium chloride and the balance of deionized water, wherein the pH value is natural, and the mixture is sterilized at 121 ℃ for 30 min;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to the ratio of 3:1, adding 1% (E/S by mass of a substrate) of enzyme, carrying out vacuum filtration at the enzymolysis temperature of 55 ℃ for 4 hours, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium of the brevibacillus laterosporus MES818 is as follows: glucose 4%, molasses 3%, yeast extract powder 1%, peanut meal 4%, sodium chloride 2%, magnesium sulfate 1%, neutral protease 0.5 ‰, flavourzyme 2 ‰, sodium citrate 3%, foam killer 0.2 ‰, and deionized water in balance, wherein the pH of culture medium is 6.0, and the culture medium is sterilized at 121 deg.C for 30 min.

Example 5

The invention also provides a composite microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae, which is prepared by fully mixing the fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 according to the volume ratio of 3:2, then centrifuging the mixture for 30min at 10000rpm to obtain thalli, and suspending and scattering the thalli by sterile water, so that the total number of the living bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

(2) preparing a seed solution: absorbing the strains stored at minus 80 ℃, inoculating the strains into 300mL of liquid culture medium according to the inoculum size of 10%, and culturing at 37 ℃ and 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with the inoculation amount of 10%, wherein the charging coefficient of the seed tank is 50%, the fermentation temperature is 37 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.8, the fermentation period is 48h, and 5M NaOH is fed during the fermentation process to control the pH value of a fermentation liquid to be 6.8;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to the inoculation amount of 10% for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 50%, the fermentation temperature is 37 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.8, the fermentation is carried out for 24 hours, exogenous nutrients (mixed liquid of 1% of glucose and 1% of soybean polypeptide) are fed in a feeding manner, the fermentation is continued for 10 hours, and after the fermentation is finished, the Bacillus pumilus MES828 liquid fermentation product can be obtained.

(1) preparing strains; transferring separated Brevibacillus laterosporus MES818 to an eggplant bottle culture medium, culturing at 37 ℃ for 72h to obtain an activated strain, adding 30ml of protective agent (2.0% glucose solution, 3.5% skimmed milk powder solution and 20% glycerol solution) into an eggplant bottle, scraping by using a sterile spatula, grinding uniformly by using a grinder, and storing to a refrigerator at-80 ℃ for later use;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a 50L seed tank for fermentation with the inoculation amount of 10%, wherein the charging coefficient of the seed tank is 45%, the fermentation temperature is 37 ℃, the rotating speed is 220rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 7.0, the fermentation period is 33h, and 5M NaOH is fed during the fermentation process to control the pH value of the fermentation liquid to be 7.0;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a 5000L fermentation tank according to 15% of inoculation amount for liquid fermentation, wherein the charging coefficient of the fermentation tank is 50%, the fermentation temperature is 37 ℃, the rotating speed is 230rpm, the tank pressure is kept between 0.05Mpa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation culture medium is 6.8, when the fermentation is carried out for 30 hours, exogenous nutrients (mixed liquid of 1% of glucose and 3% of soybean polypeptide) are fed in a feeding mode, the fermentation is continued for 11 hours, and after the fermentation is finished, a short bacillus laterosporus MES818 liquid fermentation product can be obtained.

Preferably, the eggplant bottle culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 1% of tryptone, 0.8% of beef extract, 0.5% of sodium chloride, 2% of agar and the balance of deionized water, controlling the pH value at 7.0, and sterilizing at 121 ℃ for 30 min;

the seed liquid culture medium for the Bacillus pumilus MES828 during culture specifically comprises the following components in parts by mass: fish peptone 0.8%, soybean peptone 0.8%, yeast powder 0.5%, NaCl 1%, natural pH value, sterilizing at 121 deg.C for 30 min;

the seed tank culture medium for culturing the bacillus pumilus MES828 specifically comprises the following components in parts by mass: 2% of molasses, 1% of soybean meal, 1% of enzymatic peanut meal, 0.8% of yeast extract, 1% of sodium chloride, 0.5% of magnesium sulfate, 2% of citric acid and KH₂PO₄0.08 percent of the total weight of the mixture and the balance of deionized water, controlling the pH value to be 7.0, and sterilizing for 30min at 121 ℃;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to the ratio of 3:1, adding 1% (E/S by mass of a substrate) of enzyme, carrying out enzymolysis at the temperature of 55 ℃ for 5 hours, carrying out vacuum filtration, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium of the bacillus pumilus MES828 is as follows: 2% of white sugar, 1% of molasses, 1% of peanut cake powder, 0.8% of yeast extract, 0.5% of sodium chloride, 1% of magnesium sulfate, 1% of citric acid and KH₂PO₄0.08%, 0.5% of neutral protease, 0.5% of flavourzyme, 0.5% of natural killer and the balance of deionized water, wherein the pH value of the culture medium is 6.0, and the culture medium is sterilized for 30min at 121 ℃.

Preferably, the eggplant flask culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: tryptone 0.5%, beef extract 1%, sodium chloride 0.5%, agar 2%, and deionized water in balance, controlling pH at 7.0, and sterilizing at 121 deg.C for 30 min;

the seed liquid culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: fish peptone 0.8%, soybean peptone 0.8%, yeast powder 1%, NaCl 0.5%, natural pH value, sterilizing at 121 deg.C for 30 min;

the seed tank culture medium for culturing the brevibacillus laterosporus MES818 specifically comprises the following components in percentage by mass: 0.8% of yeast extract powder, 1% of molasses, 2% of enzymatic peanut meal, 0.7% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.05% of calcium carbonate, 0.5% of sodium chloride and the balance of deionized water, wherein the pH is natural, and the materials are sterilized for 30min at 121 ℃;

the peanut meal powder subjected to enzymolysis is obtained by compounding flavourzyme and neutral protease according to the ratio of 1:1, adding 1% (E/S by mass of a substrate) of enzyme, carrying out enzymolysis at the temperature of 40 ℃ for 6 hours, carrying out vacuum filtration, and drying at the temperature of 80 ℃;

the fermentation tank liquid culture medium of the brevibacillus laterosporus MES818 is as follows: 2% of glucose, 1% of molasses, 1% of yeast extract powder, 2% of peanut meal, 1% of sodium chloride, 0.8% of magnesium sulfate, 1% of neutral protease, 2% of flavourzyme, 1% of sodium citrate, 0.2% of sodium chloride and the balance of deionized water, wherein the pH value of a culture medium is 7.0, and the culture medium is sterilized for 30min at 121 ℃.

Example 6

The application of the compound microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae family is to dilute the compound microbial inoculum by water according to the ratio of 1: 1000-1: 500, and then apply the compound microbial inoculum to the succulent plant leaves in a leaf surface spraying mode or apply the compound microbial inoculum to the roots of the succulent plants in a root irrigation mode.

The complex microbial inoculum (prepared in examples 2-5) was applied by foliar spray.

Respectively carrying out activation culture on bacillus pumilus MES828 and bacillus laterosporus MES818 to logarithmic growth phase according to the method, centrifuging the fermentation product of the activated bacillus pumilus MES828 and the fermentation product of the bacillus laterosporus MES818 at 5000-10000 rpm for 15-30 min to obtain thalli, and carrying out resuspension and scattering on the thalli by using sterile water to prepare a microbial inoculum so that the effective viable count of a single strain is 1.0 multiplied by 10⁹～1.0×10¹⁰Within the range; then mixing the fermentation product of the activated Bacillus pumilus MES828 and the fermentation product of the Bacillus laterosporus MES818 according to the volume ratio of 1-4:1-4, then centrifuging at 5000-10000 rpm for 15-30 min to obtain thalli, and then suspending and scattering the thalli by sterile water to prepare a microbial inoculum so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2×10¹⁰Within the range.

The using method comprises the following steps: after a bacillus pumilus agent, a bacillus laterosporus agent, a compound agent and 70% thiophanate methyl wettable powder are diluted by water according to the ratio of 1:500, the leaf surfaces of the bacillus pumilus agent, the bacillus laterosporus agent and the compound agent are sprayed on the leaves of the polypide plants in the Crassulaceae.

The experimental site is selected from a Vivian farm greenhouse in a Wuqing district Chengjizhen meishi highway Ponzhuang greenhouse base.

And (3) experimental design: selecting succulent plant saussurea involucrata seed seedling with basically consistent growth vigor for application effect research. The seedling raising substrate is prepared by mixing turf and vermiculite according to a ratio of 2:1, and transplanting the seed seedlings into a multi-meat flowerpot (the caliber is 17cm, and the height is 14). The test set-up is shown in table 2 below. Each treatment was set to 20 pots and repeated 3 times. The treatment solution is sprayed once every 7 days, the spraying amount is that the leaf surfaces are completely soaked in the treatment solution, the management is normal, and the disease index and the prevention and treatment effect are calculated, which are specifically shown in the following table 2.

Design of experiments

TABLE 2 comparative results of control of brown rot of succulent plants of Crassulaceae by different foliar spray treatment modes

Group of	Treatment method	Index of disease condition	Prevention effect/%)
				Test 1 group	Spraying short bacillus agent on leaf surface	16.14b	66.4
Test 2 groups	Spraying Brevibacillus laterosporus microbial inoculum on leaf surfaces	15.31b	68.9
				Test 3 groups	Foliage spraying composite bacterial agent	10.10bc	84.4
Test 4 groups	70% thiophanate methyl wettable powder sprayed on leaf surfaces	9.11c	92.6
				Control group	Spraying sterile water on leaf surfaces	85.21a	—

The experimental results are as follows: the test results in table 2 show that the disease index of the test 1 group to the Crassulaceae succulent plant brown rot is 16.14, and the control effect is 66.4%; experiment 2, the disease index of the crassula clavata of the crassulaceae is 15.31, and the control effect is 68.9%; the disease index of the test 3 group on the brown rot of the succulent plants in the crassulaceae is 10.10, and the control effect is 84.4%, and the disease index of the test 4 group on the brown rot of the succulent plants in the crassulaceae is 9.11, and the control effect is 92.6%.

Example 7

Respectively carrying out activation culture on bacillus pumilus MES828 and bacillus laterosporus MES818 to logarithmic growth phase according to the method, centrifuging the fermentation product of the activated bacillus pumilus MES828 and the fermentation product of the bacillus laterosporus MES818 at 5000-10000 rpm for 15-30 min to obtain thalli, and carrying out resuspension and scattering on the thalli by using sterile water to prepare a microbial inoculum so that the effective viable count of a single strain is 1.0 multiplied by 10⁹～1.0×10¹⁰Within the range; then mixing the fermentation product of the activated Bacillus pumilus MES828 and the fermentation product of the Bacillus laterosporus MES818 according to the volume ratio of 1-4:1-4, then centrifuging at 5000-10000 rpm for 15-30 min to obtain thalli, and then suspending and scattering the thalli by sterile water to prepare a microbial inoculum so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

The using method comprises the following steps: after a bacillus pumilus agent, a bacillus laterosporus agent, a composite agent and 70% thiophanate methyl wettable powder are diluted by water according to the ratio of 1:1000, leaf surfaces of the bacillus pumilus agent, the bacillus laterosporus agent and the composite agent are sprayed on leaves of a succulent plant of the Crassulaceae.

And (3) experimental design: selecting succulent plant saussurea involucrata seed seedling with basically consistent growth vigor for application effect research. The seedling raising substrate is prepared by mixing turf and vermiculite according to a ratio of 2:1, and transplanting the seed seedlings into a multi-meat flowerpot (the caliber is 17cm, and the height is 14). The test set-up is shown in table 2 below. Each treatment was set to 20 pots and repeated 3 times. The treatment solution is sprayed once every 10 days, the spraying amount is that the leaf surfaces are completely soaked in the treatment solution, the management is normal, and the disease index and the prevention and treatment effect are calculated, which is specifically shown in the following table 3.

Design of experiments

TABLE 3 comparative test results of control effect of different foliar spray treatment modes on brown rot of succulent plants of Crassulaceae

Group of	Treatment method	Index of disease condition	Prevention effect/%)
				Test 1 group	Spraying short bacillus agent on leaf surface	15.33b	68.8
Test 2 groups	Spraying Brevibacillus laterosporus microbial inoculum on leaf surfaces	14.97b	70.6
				Test 3 groups	Foliage spraying composite bacterial agent	10.94bc	86.8
Test 4 groups	70% thiophanate methyl wettable powder sprayed on leaf surfaces	8.96c	95.6
				Control group	Spraying sterile water on leaf surfaces	85.21a	—

The experimental results are as follows: the test results in table 3 show that the disease index of the test 1 group to the Crassulaceae succulent plant brown rot is 15.33, and the control effect is 68.8%; experiment 2, the disease index of the group of Crassulaceae succulent plants to brown rot is 14.97, and the control effect is 70.6%; the disease index of 3 groups of experiments on the brown rot of the succulent plants in the crassulaceae is 10.94, and the control effect is 86.8%, and the disease index of 4 groups of experiments on the brown rot of the succulent plants in the crassulaceae is 8.96, and the control effect is 95.6%.

Example 8

The complex microbial inoculum (prepared in examples 2-5) was applied in a pot root manner.

Respectively carrying out activation culture on bacillus pumilus MES828 and bacillus laterosporus MES818 to logarithmic growth phase according to the method, centrifuging the fermentation product of the activated bacillus pumilus MES828 and the fermentation product of the bacillus laterosporus MES818 at 5000-10000 rpm for 15-30 min to obtain thalli, and carrying out resuspension and scattering on the thalli by using sterile water to prepare a microbial inoculum so that the effective viable count of a single strain is 1.0 multiplied by 10⁹～1.0×10¹⁰Within the range; then mixing the activated fermentation product of Bacillus pumilus MES828 and the fermentation product of Bacillus laterosporus MES818 according to the volume ratio of 1-4:1-4, centrifuging at 5000-10000 rpm for 15-30 min to obtain thallus, and usingThe bacteria are re-suspended and scattered by sterile water to prepare a microbial inoculum, so that the total number of viable bacteria in a compound microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

The using method comprises the following steps: diluting Bacillus pumilus agent, Brevibacillus laterosporus agent, composite agent and 70% thiophanate methyl wettable powder with water at a ratio of 1:500, and irrigating roots of succulent plants of Crassulaceae.

And (3) experimental design: selecting succulent plant saussurea involucrata seed seedling with basically consistent growth vigor for application effect research. The seedling raising substrate is prepared by mixing turf and vermiculite according to a ratio of 2:1, and transplanting the seed seedlings into a multi-meat flowerpot (the caliber is 17cm, and the height is 14). The test set-up is shown in table 2 below. Each treatment was set to 20 pots and repeated 3 times. Irrigating 30ml of the treatment solution into each pot, irrigating the roots once every 7 days, performing normal management, and calculating disease index and prevention and treatment effect, which are shown in the following table 4.

Design of experiments

TABLE 4 comparative test results of control effect of different root irrigation treatment modes on brown rot of succulent plants of Crassulaceae

Group of	Treatment method	Index of disease condition	Prevention effect/%)
				Test 1 group	Root-irrigation bacillus pumilus microbial inoculum	15.71b	68.2
Test 2 groups	Brevibacillus aschersonia gazei preparation	14.82b	70.1
				Test 3 groups	Root-irrigation composite bacterial agent	11.10bc	84.2
Test 4 groups	Drenched 70% thiophanate methyl wettable powder	8.82c	96.2
				Control group	Root-irrigation sterile water	85.21a	—

The experimental results are as follows: the test results in table 4 show that the disease index of the test 1 group to the Crassulaceae succulent plant brown rot is 15.71, and the control effect is 68.2%; experiment 2, the disease index of the group of Crassulaceae succulent plants to brown rot is 14.82, and the control effect is 70.1%; the disease index of the crassula bicolor of crassulaceae in test 3 group is 11.10, and the control effect is 84.2%, and the disease index of the crassula bicolor of crassulaceae in test 4 group is 8.82, and the control effect is 96.2%.

Example 9

Respectively performing activation culture on Bacillus pumilus MES828 and Bacillus laterosporus MES818 according to the above methodUntil logarithmic phase, centrifuging the fermentation product of activated Bacillus pumilus MES828 and Bacillus laterosporus MES818 at 5000-10000 rpm for 15-30 min to obtain thallus, suspending and scattering the thallus with sterile water to obtain microbial inoculum, wherein the effective viable count of single strain is 1.0 × 10⁹～1.0×10¹⁰Within the range; then mixing the fermentation product of the activated Bacillus pumilus MES828 and the fermentation product of the Bacillus laterosporus MES818 according to the volume ratio of 1-4:1-4, then centrifuging at 5000-10000 rpm for 15-30 min to obtain the product, and using sterile water to suspend and scatter the thallus to prepare a microbial inoculum so that the total number of viable bacteria in the composite microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10¹⁰Within the range.

The using method comprises the following steps: diluting a bacillus pumilus agent, a bacillus laterosporus agent, a composite agent and 70% thiophanate methyl wettable powder by water according to the ratio of 1:1000, and then irrigating roots of the succulent plants in the Crassulaceae.

And (3) experimental design: selecting succulent plant saussurea involucrata seed seedling with basically consistent growth vigor for application effect research. The seedling raising substrate is prepared by mixing turf and vermiculite according to a ratio of 2:1, and transplanting the seed seedlings into a multi-meat flowerpot (the caliber is 17cm, and the height is 14). The test set-up is shown in table 2 below. Each treatment was set to 20 pots and repeated 3 times. Irrigating 30ml of the treatment solution into each pot, irrigating the roots once every 10 days, performing normal management, and calculating disease indexes and prevention and treatment effects, which are shown in the following table 5.

Design of experiments

TABLE 5 comparative test results of control effect of different root irrigation treatment modes on brown rot of succulent plants of Crassulaceae

Group of	Treatment method	Index of disease condition	Prevention effect/%)
				Test 1 group	Root-irrigation bacillus pumilus microbial inoculum	15.21b	74.8
Test 2 groups	Brevibacillus aschersonia gazei preparation	14.71b	75.2
				Test 3 groups	Root-irrigation composite bacterial agent	10.11bc	88.3
Test 4 groups	Drenched 70% thiophanate methyl wettable powder	8.71c	96.4
				Control group	Root-irrigation sterile water	85.21a	—

The experimental results are as follows: the test results in table 5 show that the disease index of the test 1 group to the Crassulaceae succulent plant brown rot is 15.21, and the control effect is 74.8%; experiment 2 group has disease index of Crassulaceae succulent plant brown rot of 14.71, and control effect is 75.2%; the disease index of the test 3 group on the brown rot of the succulent plants in the crassulaceae is 10.11, and the control effect is 88.3%, and the disease index of the test 4 group on the brown rot of the succulent plants in the crassulaceae is 8.71, and the control effect is 96.4%.

The test results of the examples 6-9 show that the control effect of the composite microbial inoculum is slightly inferior to that of the chemical agent. But long-term application of chemical agents is unfavorable for the growth of plants, and simultaneously influences the structure of rhizosphere microbial flora of the plants, and the complex microbial inoculum has the characteristics of no toxicity, no harm, no residue, no inhibition on growth and small dosage; the composite microbial inoculum for preventing and treating brown rot of the polypide of Crassulaceae can effectively prevent and treat brown rot of the polypide of Crassulaceae, can promote nutrient absorption and utilization of the polypide of Crassulaceae, improves the form of the polypide of Crassulaceae, and has important positive significance for enhancing the ornamental value of the polypide of Crassulaceae. Compared with the single application of the bacillus pumilus and the bacillus brevis, the composite microbial inoculum has stronger antagonistic action, and the two composite strains are mutually synergistic, can be planted, propagated and transferred in rhizosphere, root surface and body of the polypide of the crassulaceae, and has stronger antagonistic effect on brown rot of the polypide of the scenaceae. Therefore, the compound microbial inoculum is preferably selected for preventing and treating the brown rot of the succulent plants in the Crassulaceae family.

SEQUENCE LISTING

<110> Kunzea biotechnology Limited in Tianjin development area

<120> composite microbial inoculum for preventing and treating succulent plant brown rot, and preparation method and application thereof

<140> 201811628263.1

<160> 2

<210> 1

<211> 1453

<212> DNA

<213> Bacillus Pumilus

<400> 1

gcgggggggg gctatactgc agtcgagcgg acagaaggga gcttgctccc ggatgttagc 60

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120

ccggagctaa taccggatag ttccttgaac cgcatggttc aaggatgaaa gacggtttcg 180

gctgtcactt acagatggac ccgcggcgca ttagctagtt ggtggggtaa tggctcacca 240

aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300

ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360

gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420

aagtgcgaga gtaactgctc gcaccttgac ggtacctaac cagaaagcca cggctaacta 480

cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 540

agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt 600

cattggaaac tgggaaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg 660

aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga 720

cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780

aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 840

agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc 900

gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960

gacatcctct gacaacccta gagatagggc tttcccttcg gggacagagt gacaggtggt 1020

gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080

ccttgatctt agttgccagc atttagttgg gcactctaag gtgactgccg gtgacaaacc 1140

ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200

ctacaatgga cagaacaaag ggctgcgaga ccgcaaggtt tagcgaatcc cataaatctg 1260

ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1320

cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacaa 1380

tacacgagag tttgcaacac ccgaagtcgg tgaggtaacc tttatggagc cagccgccga 1440

agctgacaga gag 1453

<210> 2

<211> 1437

<212> DNA

<213> Brevibacillus laterosporu

<400> 1

gtaggggggg gtctataatg cagtcgagcg agggttttcg gaccctagcg gcggacgggt 60

gagtaacacg taggcaacct gcctgtaaga ctgggataac atagggaaac ttatgctaat 120

accggataga gttttgcttc gcatgaagcg aaacggaaag atggcgcaag ctatcacttg 180

cagatgggcc tgcggcgcat tagctagttg gtgaggtaaa ggctcaccaa ggcgacgatg 240

cgtagccgac ctgagagggt gaccggccac actgggactg agacacggcc cagactccta 300

cgggaggcag cagtagggaa ttttccacaa tggacgaaag tctgatggag caacgccgcg 360

tgaacgatga aggctttcgg gtcgtaaagt tctgttgtta gggaagaaac agtgccattt 420

aaataaggtg gcaccttgac ggtacctaac gagaaagcca cggctaacta cgtgccagca 480

gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgca 540

ggtggctatg taagtctgat gttaaagccc ggggctcaac ctcggttcgc attggaaact 600

gcgtagcttg agtgcaggag aggaaagtgg tattccacgt gtagcggtga aatgcgtaga 660

gatgtggagg aacaccagtg gcgaaggcga ctttctggcc tgtaactgac actgaggcgc 720

gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780

gctaggtgtt aggggtttca atacccttag tgccgcagct aacgcaataa gcactccgcc 840

tggggagtac gctcgcaaga gtgaaactca aaggaattga cgggggcccg cacaagcggt 900

ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcccact 960

gaccgctcta gagatagagc ttcccttcgg ggcagtggtg acaggtggtg catggttgtc 1020

gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttatcttta 1080

gttgccagca ttcagttggg cactctagag agactgccgt cgacaagacg gaggaaggcg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggtt 1200

ggtacaacgg gatgctactt cgcgagaaga tgctaatctc ttaaaaccaa tctcagttcg 1260

gattgtaggc tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat 1320

gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gggagtttgc 1380

aacacccgaa gtcggtgagg taaccgcaag gagccagccg ccgaaggtgg agatccg 1437

Claims

1. The compound microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae is characterized by comprising a fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and a fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818, wherein the volume ratio of the Bacillus Pumilus (Bacillus Pumilus) MES828 to the fermentation product of the Bacillus laterosporus (Brevibacillus laterosporus) MES818 is 1-4:1-4, and the total number of living bacteria in the compound microbial inoculum product is 2.5 multiplied by 10⁹～2.0×10 ¹⁰Within the range;

the Bacillus Pumilus (Bacillus Pumilus) MES828 is preserved in China general microbiological culture collection management center, the preservation address is No. 3 of Xilu No.1 of Beijing Korean-Yang district, the preservation date is 2018, 12 and 03 days, and the preservation number is CGMCC No. 16858;

2. The preparation method of the compound microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae family as claimed in claim 1, wherein the fermentation products of Bacillus Pumilus (Bacillus Pumilus) MES828 and Bacillus laterosporus (Brevibacillus laterosporus) MES818 are prepared by liquid high-density fermentation technology, wherein the viable count of the fermentation products of Bacillus Pumilus (Bacillus Pumilus) MES828 is not less than 2.5 x 10⁹cFu/g, the number of viable bacteria in the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 is not less than 2.5 × 10⁹ cFu/g。

3. The preparation method of the compound microbial inoculum for preventing and treating the brown rot of the succulent plants of the Crassulaceae family as claimed in claim 2, wherein the fermentation product of Bacillus Pumilus (Bacillus Pumilus) MES828 and the fermentation product of Bacillus laterosporus (Brevibacillus laterosporus) MES818 are fully mixed according to the volume ratio of 1-4:1-4, and then centrifuged at 5000-10000 rpm for 15-30 min to obtain thalli, and the thalli is resuspended and scattered by sterile water to prepare the compound microbial inoculum, so that the total number of viable bacteria in the compound microbial inoculum product is 2.5 x 10⁹ ～2.0×10¹⁰Within the range.

4. The preparation method of the composite microbial inoculum for preventing and treating the brown rot of the succulent plants in the Crassulaceae family as claimed in claim 2, wherein the high-density fermentation method of the Bacillus pumilus comprises the following steps:

(1) preparing strains; transferring separated Bacillus Pumilus (Bacillus Pumilus) MES828 to an eggplant bottle culture medium, culturing at 35-37 ℃ for 48-72 h to obtain an activated strain, and adding a protective agent for storage for later use;

(2) preparing a seed solution: inoculating the seed into a liquid culture medium according to the inoculation amount of 5-15%, and culturing at 35-37 ℃ at 220r/min for 72h to obtain seed liquid;

(3) fermenting in a seeding tank: inoculating the prepared seed liquid into a seed tank by an inoculation amount of 5-15% for fermentation, wherein the charging coefficient of the seed tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotating speed is 220-250 rpm, the tank pressure is kept between 0.05-0.08 MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a seed culture medium is 6.5-7.0, the fermentation period is 24-36 h, and 5-10M NaOH is fed during the fermentation process to control the pH value of a fermentation liquid to be 6.5-7.0;

(4) fermentation in a fermentation tank: after the fermentation of the seeding tank is finished, transferring the seeds to a fermentation tank according to the inoculation amount of 5-15% for deep liquid fermentation, wherein the charging coefficient of the fermentation tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotating speed is 220-250 rpm, the tank pressure is kept between 0.05-0.08 MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation medium is 6.5-7.0, when the fermentation is carried out for 24-36 h, an external nutrient is added in a feeding mode, the fermentation is continued for 10-12 h, and after the fermentation is finished, a Bacillus pumilus MES828 liquid fermentation product can be obtained.

5. The preparation method of the complex microbial inoculant for preventing and treating the brown rot of succulent plants as claimed in claim 2, wherein the high-density fermentation method of brevibacillus laterosporus comprises the following steps:

(1) preparing strains; transferring separated Brevibacillus laterosporus MES818 to an eggplant bottle culture medium, culturing at 35-37 ℃ for 48-72 h to obtain an activated strain, and adding a protective agent for storage for later use;

(2) preparing a seed solution: inoculating the seed into 300mL of liquid culture medium according to the inoculation amount of 5-15%, and culturing at 35-37 ℃ at 220r/min for 72h to obtain seed liquid;

(4) fermentation in a fermentation tank: after the fermentation of the seed tank is finished, transferring the seeds to a fermentation tank according to the inoculation amount of 5-15% for liquid fermentation, wherein the charging coefficient of the fermentation tank is 45-55%, the fermentation temperature is 35-37 ℃, the rotating speed is 220-250 rpm, the tank pressure is kept between 0.05-0.08 MPa, the ventilation ratio is 1: 0.5-1, the initial pH value of a fermentation medium is 6.5-7.0, when the fermentation is carried out for 24-36 h, exogenous nutrients are fed in a feeding mode, the fermentation is continued for 10-12 h, and after the fermentation is finished, a short bacillus laterosporus MES818 liquid fermentation product can be obtained.

6. The preparation method of the complex microbial inoculant for preventing and treating the brown rot of succulent plants as claimed in claim 3, wherein the eggplant bottle culture medium components in the Bacillus pumilus MES828 culture are as follows by mass: 0.5-2% of tryptone, 0.5-2% of beef extract, 0.2-1% of sodium chloride, 1.5-2% of agar and the balance of deionized water, controlling the pH value to be 7.0-7.3, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium components of the Bacillus pumilus MES828 during culture are as follows by mass: 0.5-2% of fish peptone, 0.5-2% of soybean peptone, 0.5-2% of yeast powder, 0.2-1% of NaCl, natural pH value and sterilization at 121 ℃ for 30 min;

the seed tank culture medium components of the Bacillus pumilus MES828 during culture are as follows by mass: 2 to 4 percent of molasses, 0.5 to 3 percent of soybean meal, 0.5 to 2 percent of enzymolysis peanut meal, 0.5 to 1 percent of yeast extract, 0.2 to 2 percent of sodium chloride, 0.5 to 2 thousandths of magnesium sulfate, 0.2 to 2 percent of citric acid, 40.05 to 0.10 percent of KH 2 PO and the balance of deionized water, wherein the pH value is controlled to be 7.0 to 7.3, and the sterilization is carried out for 30min at the temperature of 121 ℃;

the enzymatic peanut meal is prepared by compounding flavourzyme and neutral protease according to the ratio of 1: 3-3: 1, wherein the enzyme adding amount is 0.5-1% by mass of a substrate; the enzymolysis temperature is 40-55 ℃, the enzymolysis time is 4-6 h, and the product is obtained after vacuum filtration and drying at 80 ℃;

the components of the fermentation tank liquid culture medium during the culture of the bacillus pumilus MES828 are as follows by mass fraction: 1-3% of white sugar, 1-3% of molasses, 0.5-2% of peanut cake powder, 0.5-1% of yeast extract, 0.2-2% of sodium chloride, 0.5-2% of magnesium sulfate, 0.2-2% of citric acid, 40.05-0.1% of KH 2 PO, 0.5-2% of neutral protease, 0.5-2% of flavourzyme, 0.2-1.5% of natural enemy, the balance of deionized water, the pH value of a culture medium is 6.0-7.0, and the culture medium is sterilized for 30min at 121 ℃.

7. The preparation method of the complex microbial inoculant for preventing and treating the brown rot of succulent plants as claimed in claim 4, wherein the ingredients of the culture medium of the eggplant flask during the culture of the Brevibacillus laterosporus MES818 are as follows by mass fraction: 0.5-2% of tryptone, 0.5-2% of beef extract, 0.2-1% of sodium chloride, 1.5-2% of agar and the balance of deionized water, controlling the pH value to be 7.0-7.3, and sterilizing for 30min at 121 ℃;

the seed liquid culture medium components of the brevibacillus laterosporus MES818 during the culture are specifically as follows according to the mass fraction: 0.5-2% of fish peptone, 0.5-2% of soybean peptone, 0.5-2% of yeast powder, 0.2-1% of NaCl, natural pH value and sterilization at 121 ℃ for 30 min;

the components of a seed tank culture medium in the process of culturing Brevibacillus laterosporus MES818 are as follows by mass fraction: 0.5-2% of yeast extract powder, 1-3% of molasses, 1-4% of enzymatic hydrolysis peanut meal powder, 0.5-1% of monopotassium phosphate, 0.03-0.05% of magnesium sulfate, 0.03-0.05% of calcium carbonate, 0.5-1% of sodium chloride and the balance of deionized water, wherein the pH is natural, and the mixture is sterilized at 121 ℃ for 30 min;

the components of the fermentation tank liquid culture medium in the process of culturing the brevibacillus laterosporus MES818 are specifically as follows by mass fraction: 2-4% of glucose, 1-3% of molasses, 1-3% of yeast extract powder, 2-4% of peanut meal, 1-3% of sodium chloride, 1-2% of magnesium sulfate, 0.5-2% of neutral protease, 0.5-2% of flavourzyme, 0.5-3% of sodium citrate, 0.2-1.5% of foam killer and the balance of deionized water, wherein the pH value of the culture medium is 6.0-7.0, and the culture medium is sterilized at 121 ℃ for 30 min.

8. The application of the complex microbial inoculant for preventing and treating the brown rot of the succulent plants as claimed in claim 1, wherein the complex microbial inoculant is diluted by water according to a ratio of 1: 1000-1: 500 and then applied to the succulent plant leaves in a leaf spraying manner or applied to the roots of the succulent plants in a root irrigation manner.